Cytokine signaling involves a family of transcription factors known as Signal Transducer and Activators of Transcription (Stat) that are activated by receptor-bound tyrosine kinases of the Janus Kinase (Jak) family, discovered and characterized during the earlier years of this project. Stat1 is activated mainly by the interferon (IFN) system but has additional roles outside the IFN system, while its closest relative, Stat3, is a common target for many cytokine receptors. Stat3 is also activated by classical growth factor receptor tyrosine kinases and by cytoplasmic proto-oncogene tyrosine kinases and their oncogenic derivatives. Stat3 function is essential during early embryogenesis, but its role in later development and in mature animals remains to be determined. Stat2 is activated exclusively by IFNalpha and is the most potent transcriptional activator of the Stat family. This proposal will take a genetic and biochemical approach to continued studies of Stat signaling and function. We have previously created animals devoid of Stat1 and will continue our studies of the resulting phenotype, concentrating on the mechanisms underlying newly discovered IFN- independent actions of Stat1. These include a constitutive role in basal gene expression as well as a role in chondrocyte growth and bone development. Structure/function analysis of Stat1 will examine its role in gene expression, and the ability of a Stat1 mutation to suppress bone abnormalities caused by FGF will be determined. By creating cell lines and animals that carry a conditionally null mutation of the Stat3 gene, we will ablate Stat3 function at specific times of development, in specific tissues, and in cultured cell lines, and measure a variety of responses in its absence. We will assess the ability of Stat3-null cells to contribute to different tissues of the embryo and adult mouse. Using embryonic fibroblasts, we will determine the role of Stat3 in growth factor, cytokine, and IFN signaling responses, in cell proliferation, and in transformation in response to tyrosine kinase oncogenes. We will also assess the role of Stat3 during normal cell cycle progression, growth, and apoptosis. As a transcription factor, Stat3 dimerizes with Stat1, but the consequences of this cooperation are unknown. Regulation of Stat target genes will be measured in the presence and absence of Stat1 and Stat3. Mechanisms of transcriptional transactivation by Stat2 will be characterized by structure/function analysis of human and mouse homologues, and the role of histone acetylases will be evaluated. Results from these studies will enhance our understanding of cytokine networks, cooperating transcription factors, cell growth, and oncogenic transformation.